This competing renewal application (R01 AG027678) will extend and expand on research that was responsive to RFA-AG-05-008 Biology of the Perimenopause: Impact on Health and Aging. Findings from the first award period demonstrate that: 1) endothelial function (endothelium-dependent dilation; EDD) becomes more impaired with declining ovarian function, and 2) oxidative stress is one of the underlying mechanisms involved in impaired EDD. The global aim of this renewal application is to determine the role of tetrahydrobiopterin (BH4; an essential cofactor for endothelial nitric oxide synthase (eNOS) and nitric oxide [NO] synthesis) and eNOS uncoupling as potential mediators of oxidative stress-related endothelial dysfunction with the menopause transition and aging in women. This will be assessed in Aim 1 using cross-sectional comparisons of healthy pre-, peri- and postmenopausal women. Endothelial function will be measured under basal conditions and following an acute increase in BH4. Aim 2 will expand on the cross-sectional comparisons by using short-term (10 days) suppression of estrogen (E2) using gonadotropin releasing hormone antagonist [GnRHant] in pre-and perimenopausal women to determine the age-independent effects of E2 on EDD. Aim 3 will examine the co-administration of BH4 and an anti-oxidant (ascorbic acid; AA) for the restoration of EDD. The primary hypotheses are that the reduced EDD in peri- and postmenopausal women, and in pre- and perimenopausal women following short-term E2 suppression, will increase in response to oral BH4. Secondary hypotheses predict that the reduced EDD in peri- and postmenopausal women (at baseline and with E2 suppression) will be associated with plasma and endothelial cell protein markers of BH4 biosynthesis and oxidative stress in vascular endothelial cells. A tertiary hypothesis is that the co-administration of BH4 an AA will restore EDD in peri-and postmenopausal women to levels of premenopausal women. To test these hypotheses, brachial artery EDD will be measured in: 1) pre-and early perimenopausal women before and after short-term E2 suppression (GnRHant) and add-back of either transdermal E2 or placebo; and 2) postmenopausal women at baseline. To determine possible mechanisms for endothelial dysfunction, EDD will also be measured after oral BH4 supplementation alone and during AA infusion. Insight into the molecular events underlying the decrease in EDD will be determined by assessing the changes in endothelial cell expression of proteins involved in the regulation of cellular and systemic adaptations to E2 deficiency including BH4 biosynthesis and oxidative stress. [The results from this research should expand our earlier findings and elucidate further the mechanisms that mediate endothelial dysfunction across the stages of the menopause transition and whether these processes are triggered by E2 deficiency. Understanding these mechanistic defects will help to inform the critical window of intervention and guide future sex-specific interventions and therapies for the maintenance of vascular function and prevention of future cardiovascular diseases.]